Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Response to Amendment
Applicant’s amendment filed on 03/12/2026 has been entered. Claim(s) 10, 22, and 35 is/are cancelled. Claims 1-6, 8, 9, 11-20, 23-33, and 36 are pending and examined herein.
Status of Rejection
Applicant’s remark and amendment have overcome each and every 112(b) rejection set forth in Office Action mailed on 12/16/2025. The 112(b) rejection of claims 25 and 26 is withdrawn in light of Applicant’s amendment.
The rejection of claims 10, 22, and 35 is obviated by Applicant’s cancellation.
The amendment necessitates new ground of rejection in view of Yan (Ultrasensitive Characterization of Charge Heterogeneity of Therapeutic Monoclonal Antibodies Using Strong Cation Exchange Chromatography Coupled to Native Mass Spectrometry, 2018).
Response to Arguments
Applicant’s arguments, see Pages 7-11, filed 03/12/2026, with respect to the rejection(s) of claim(s) 1-6, 8, 9, 11-20, 23-33, and 36 under 103 have been fully considered and are persuasive. Therefore, the rejection has been withdrawn. However, upon further consideration, a new ground(s) of rejection is made in view of Yan.
The applicant’s amendment is directed to element of requiring charge variants to be a succinimide, which is an amended matter that has not been previously considered. Applicant argues one of ordinary skill in the art would not have considered Bardotti’s ammonium salt to cure the deficiency of the primary reference Ponniah (Page 9, para. 2). Applicant states that Bardotti uses a basic eluent for anion exchange, which would have changed the structure of succinimide in a high pH environment. The additional and new reference Yan cures the deficiency of Bardotti (and subsequently Ponniah) by introducing a method of using ammonium salt in CEX-MS (See rejection below), which can be repurpose/reconfigured for AEX-MS (Yan, conclusion). Specifically, the method of Yan is directed toward mAbs molecules with pI ranges from 6.3 to 9.0, which overlaps with the pI values of the claimed invention. Furthermore, Yan discloses a combination of optimizing the pH and salt-based ammonium gradient would have enhanced separation and data quality while maintaining the native state of the molecule (In addition, the mobile phase composition and gradient were optimized to accommodate both pH and salt concentration changes to achieve efficient separation of the charge variants while keeping the pH (5.6–7.4) and salt concentration (20–150 mM) in a range where mAbs are maintained in native states. Results and Discussion, para. 1).
Claim Rejections - 35 USC § 103
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claim(s) 1-6, 8, 10, and 12-13 is/are rejected under 35 U.S.C. 103 as being unpatentable over Ponniah (Characterization of charge variants of a monoclonal antibody using weak anion exchange chromatography at subunit levels, 2016) in view of Yan (Ultrasensitive Characterization of Charge Heterogeneity of Therapeutic Monoclonal Antibodies Using Strong Cation Exchange Chromatography Coupled to Native Mass Spectrometry, 2018) and Bardotti (US 20100219335 A1) as cited in previous Office Action.
Regarding claim 1, Ponniah discloses a method for characterizing at least one charge variant of a protein of interest (An efficient strategy to characterize recombinant monoclonal antibody charge variants was established using weak anion exchange chromatography, LC-MS and IdeS digestion; Abstract), comprising:
loading a sample (The recombinant monoclonal IgG2/4 hybrid antibody (pI of peaks between 6.0 and 7.0; 1.1. Materials, pg. 50) having a protein of interest and at least one charge variant (intact antibody and its fragments after IdeS digestion, 1.2. Weak anion exchange chromatography and fraction collections, para. 3, pg. 50) of said protein of interest (The same instrument, column and mobile phases were used for the analysis of the antibody after IdeS digestion with a slightly modified gradient. 1.2. Weak anion exchange chromatography and fraction collections, para. 2, pg. 50) to an anion-exchange chromatography (AEX) column (A Waters Alliance HPLC and a weak anion exchange (WAX) column (ProPac WAX-10, 4.6 250 mm, Thermo Scientific, Sunnyvale, CA) were used for separation of antibody charge variants, 1.2. Weak anion exchange chromatography and fraction collections, para. 1, pg. 50) coupled to mass spectrometry (Under broadest reasonable interpretation, the claim limitation simply requires mass spectrometry to be conducted immediately after and does not require direct coupling; 2.2.2. Analysis of the collected fractions, pg. 51-55);
applying an increasing salt concentration gradient (Mobile phase B contained 10 mM Tris and 500 mM sodium chloride; 1.2. Weak anion exchange chromatography and fraction collections, para. 1, pg. 50) to the loaded anion exchange column to obtain an eluate (Samples were injected at 0% mobile phase B, after 5 min, mobile phase B was increased to 60% in 60 min. The column was then washed using 100% mobile phase B and then equilibrated using 0% mobile phase B for 10 min. 1.2. Weak anion exchange chromatography and fraction collections, para. 2, pg. 50);
collecting at least one fraction from the eluate of b) (Fractions from analysis of both the intact antibody and its fragments after IdeS digestion were collected; 1.2. Weak anion exchange chromatography and fraction collections, para. 3, pg. 50); and
analyzing the at least one fraction to mass spectrometry analysis to characterize said at least one charge variant of said protein of interest (2.2.2. Analysis of the collected fractions, pg. 51-55).
Ponniah discloses the protein of interest has a pI value ranges from 6.0-7.0 (The recombinant monoclonal IgG2/4 hybrid antibody (pI of peaks between 6.0 and 7.0; 1.1. Materials, pg. 50), and the charge variant is succinimide (1.5 Trypsin and Lys-C digestion, para. 3, page 50)
Ponniah teaches the range of 6.0-7.0, which overlaps with the claimed range of greater than 6.2 and 7.0. It would have been obvious to one of ordinary skill in the art before the effective filing date of the invention to have selected the overlapping portion of the ranges disclosed by the reference because selection of overlapping portion of ranges has been held to be a prima facie case of obviousness. See MPEP § 2144.05.I.
Ponniah does not explicitly disclose the salt is ammonium salt nor Ponniah discloses the ion exchange is coupled on-line to mass spectrometry. In an analogous art, Bardotti discloses LC-MS analysis using ionic eluent comprising ionic salt, in particular ammonium salt (Abstract). Bardotti discloses that a particular problem eluting analyte with an ionic eluent such as sodium buffer is that when coupled with MS, the sodim eluents can cause baseline noise and spiking in the mass spectrum (Bardotti, para. [0007]). Bardotti suggests using ionic eluent comprising ammonium salt can provide clean mass spectra and overcome the issue of sodium buffer (para. [0008]).
In another analogous art, Yan discloses a cation-exchange chromatography coupled to native mass spectrometry (Abstract) that can be reconfigured/repurposed for an anion-exchange coupled to mass spectrometry (Finally, this method may provide opportunities for further development of IEX-MS-based analysis of proteins, such as coupling of anion exchange chromatography (AEX) with online MS detection under similar conditions. This newly developed, versatile, and ultrasensitive native SCX-MS method represents a significant advancement for mAb charge heterogeneity analysis and therefore making it a valuable addition to our assay portfolio to support therapeutic mAb characterization. Conclusion). Yan discloses using conventional nonvolatile IEX buffers for direct mass spectrometry may be incompatible (Page 13014, left col., para. 2) and a strategy to address the issue is utilizing volatile salt (Page 13014, left col., para. 2).
Yan cited several instances of using ammonium-based salt/solution as pH and salt gradient for characterization of IdeS digestion (Leblanc et al. (14) reported a strong cation exchange (SCX)-MS method utilizing an ammonium formate- and ammonium acetate-based pH and salt gradient for characterization of IdeS digested mAbs. Another report by Muneeruddin et al. (15) described the use of an ammonium acetate-based salt gradient for WCX-MS analysis of proteins of molecular weights below ∼30 kDa. All of these previously reported IEX-MS methods showed successful separation and mass spectrometric detection; Page 13014, left col., para. 2). Yan specifically uses ammonium acetate and ammonium bicarbonate in their own experiment (Experimental Section). Yan specifies the need to optimize the pH and salt concentration changes to achieve efficient separation of the charge variant while keeping allowing the mAbs to maintain their native states (In addition, the mobile phase composition and gradient were optimized to accommodate both pH and salt concentration changes to achieve efficient separation of the charge variants while keeping the pH (5.6–7.4) and salt concentration (20–150 mM) in a range where mAbs are maintained in native states. Results and Discussion, para. 1).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date to have derived an AEX-MS and replaced the sodium salt of Ponniah with volatile ammonium salt as suggested by Bardotti and Yan. Doing so reduces baseline noise and spiking in the mass spectra as a result of coupling (Bardotti, para. [0007]-[0008]) while also maintaining the native state of the charge variants from pH changes (Yan, Results and Discussion, para. 1).
Regarding claim 2-3, Modified Ponniah discloses the claimed invention as discussed above in claim 1. Ponniah discloses the salt gradient is an elution gradient and ranges from about 0 mM to 300 mM sodium chloride (Mobile phase B contained 500 mM sodium chloride, and 60% mobile phase B is 300 mM sodium chloride; Samples were injected at 0% mobile phase B, after 5 min, mobile phase B was increased to 60% in 60 min. 1.2. Weak anion exchange chromatography and fraction collections, para. 2, pg. 50).
Ponniah teaches the range of 0 mM to 300 mM, which overlaps with the claimed range of 10 to 300 mM. It would have been obvious to one of ordinary skill in the art before the effective filing date of the invention to have selected the overlapping portion of the ranges disclosed by the reference because selection of overlapping portion of ranges has been held to be a prima facie case of obviousness. See MPEP § 2144.05.I.
Regarding claim 4, Modified Ponniah discloses the claimed invention as discussed above in claim 1. Ponniah (after substitution of ammonium salt) discloses the increasing salt concentration gradient applied is linear (After 5 min, a linear gradient from 10% mobile phase B to 60% mobile phase B within 50 min was applied to elute antibody variants from the column… The same instrument, column and mobile phases were used for the analysis of the antibody after IdeS digestion with a slightly modified gradient. 1.2. Weak anion exchange chromatography and fraction collections, para. 2-3, pg. 50) (Note: It is presumed linear gradient is also applied to the analysis of the antibody after IdeS digestion).
Regarding claim 5, Modified Ponniah discloses the claimed invention as discussed above in claim 3. Ponniah discloses the increasing salt concentration gradient applied is linear (After 5 min, a linear gradient from 10% mobile phase B to 60% mobile phase B within 50 min was applied to elute antibody variants from the column… The same instrument, column and mobile phases were used for the analysis of the antibody after IdeS digestion with a slightly modified gradient. 1.2. Weak anion exchange chromatography and fraction collections, para. 2-3, pg. 50) (Note: It is presumed linear gradient is also applied to the analysis of the antibody after IdeS digestion).
Regarding claim 6, Modified Ponniah discloses the claimed invention as discussed above in claim 1. Ponniah discloses the method further comprising monitoring the eluate of b) for ultraviolet absorbance (The proteins eluting off the column were monitored using UV280 nm…Protein concentrations were determined using UV absorbance at 280 nm; 1.2. Weak anion exchange chromatography and fraction collections, para. 1 and 3, pg. 50) and collecting said at least one fraction that is eluted prior to or after elution of the protein of interest (Fractions from analysis of both the intact antibody and its fragments after IdeS digestion were collected; 1.2. Weak anion exchange chromatography and fraction collections, para. 3, pg. 50).
Regarding claim 8, Modified Ponniah discloses the claimed invention as discussed above in claim 1. Ponniah discloses the protein of interest is an IgG4-based monoclonal antibody (The recombinant monoclonal IgG2/4 hybrid antibody; 1.1. Materials, pg. 50).
Regarding claim 12-13, Modified Ponniah discloses the method further comprising treating the sample prior to loading the sample on AEX column, wherein the digestion conditions include use of IdeS or variant thereofs (The same instrument, column and mobile phases were used for the analysis of the antibody after IdeS digestion with a slightly modified gradient. 1.2. Weak anion exchange chromatography and fraction collections, para. 3, pg. 50).
Claim(s) 9 is/are rejected under 35 U.S.C. 103 as being unpatentable over Ponniah in view of Bardotti and Yan as discussed above in claim 1, and further in view of Yang (Analysis and purification of IgG4 bispecific antibodies by a mixed-mode chromatography, 2015) as cited in previous Office Action.
Regarding claim 9, Modified Ponniah discloses the claimed invention as discussed above in claim 1. Ponniah does not disclose the IgG4-based monoclonal antibody (See claim 8) is bispecific.
IgG4 is known to have a bispecific antibody variant. In an analogous art, Yang discloses analysis and purification of IgG4 bispecific antibody with chromatography (Abstract). The bispecific body were derived from Chinese hamster ovary (CHO) cell lines (Yang, General, para. 1, pg. 174) (the antibody used in Ponniah is also derived from CHO cell line, see Ponniah, 1.1. Materials, pg. 50) and were separated and purified by anion exchange chromatography (Yang, General, para. 1, pg. 174).
It would have been obvious to one of ordinary skill in the art before the effective filing date to have analyzed a IgG4-like bispecific antibody using the method of Ponniah with a reasonable expectation of success. Doing so allows for studies of half molecule exchange (Abstract; LC-MS analysis of antibodies from half molecule exchange in vitro, pg. 174-175, Yang).
Claim(s) 11 is/are rejected under 35 U.S.C. 103 as being unpatentable over Ponniah in view of Bardotti and Yan as discussed above in claim 1, and further in view of Bobaly (Protocols for the analytical characterization of therapeutic monoclonal antibodies. II – Enzymatic and chemical sample preparation, 2017) and Promega (Certificate of Analysis, 2017) as cited in previous Office Action.
Regarding claim 11, Modified Ponniah discloses the claimed invention as discussed above in claim 1. Neither Ponniah, Yan, nor Bardotti discloses the method further comprising removing Fc N-glycosylation from said protein of interest prior to loading said sample on AEX column.
In an analogous art, Bobaly discloses a list of enzymatic and chemical sample preparation protocols for monoclonal antibodies (mAbs) for analytical characterization. Bobaly discloses that techniques such as N-deglycosylation (same as removing N-glycosylation) using enzymes such as PNGase F (2.3. Introduction to N-deglycosylation, pg. 330-331) simplifies the sample and thus make the sample more suitable for chromatographic and mass spectrometric analysis (The analytical characterization of therapeutic monoclonal antibodies and related proteins usually incorporates various sample preparation methodologies. Indeed, quantitative and qualitative information can be enhanced by simplifying the sample, thanks to the removal of sources of heterogeneity (e.g. N-glycans) and/or by decreasing the molecular size of the tested protein by enzymatic or chemical fragmentation. These approaches make the sample more suitable for chromatographic and mass spectrometric analysis. Abstract).
In another analogous art, Promega discloses IdeS protease can be used with PNGase F within the same reaction to perform fragmentation and removal of Fc glycan in a single step (IdeS Protease can be used in the same reaction as PNGase F (Cat.# V4831) to perform fragmentation and removal of Fc glycans in a single step using the recommended digest buffer. Ten units of PNGase F may be sufficient to remove Fc glycans from 50µg of IgG in 2 hours at 37°C, although some optimization of PNGase F amount and incubation time may be required. Usage Information, page 2, Notes, bullet point 9, left column)
It would have been obvious to one of ordinary skill in the art to have incorporated a sample preparation step of N-deglycosylation to the method of Modified Ponniah as suggested by Bobaly and Promega using PNGase F enzyme in a one pot reaction. One of ordinary skill in the art would be motivated to do so in order to improve mass spectrometry and chromatography results by simplifying the sample as disclosed by Bobaly (Abstract).
Claim(s) 14 is/are rejected under 35 U.S.C. 103 as being unpatentable over Ponniah in view of Bardotti and Yan as discussed above in claim 1, and further in view of Bailey (Charge variant native mass spectrometry benefits mass precision and dynamic range of monoclonal antibody intact mass analysis, 2018) as cited in previous Office Action.
Regarding claim 14, Modified Ponniah discloses the claimed invention as discussed above in claim 1. Neither Ponniah, Yan, nor Bardotti explicitly discloses running a mass spectrometry under native condition.
In an analogous art, Bailey discloses a procedure to perform the analysis of monoclonal antibodies using native MS (charge variant native mass spectrometry) for characterization of variants such as deamidation (Characterization of variants such as deamidation, which are traditionally unattainable by intact mass due to their minimal molecular weight differences, were measured unambiguously by mass and retention time to allow confident MS1 identification. We demonstrate that efficient chromatographic separation allows introduction of the purified forms of the charge variant isoforms into the Orbitrap mass spectrometer. Abstract).
It would have been obvious to one of ordinary skill in the art before the effective filing date to used Orbitrap MS system (which comes with native MS setting) disclosed by Bailey to the method of Ponniah. Doing so increase baseline resolution for measurement of complex biological compound such as mAbs (Introduction, para. 2, pg. 1214, Bailey).
Claim(s) 15-20, 23-24, 26, and 28-33, and 36 is/are rejected under 35 U.S.C. 103 as being unpatentable over Ponniah in view of Yan, Bardotti, Bobaly, and Promega.
Regarding claim 15, Ponniah discloses a method of characterizing at least one charge variant of a protein of interest comprising (An efficient strategy to characterize recombinant monoclonal antibody charge variants was established using weak anion exchange chromatography, LC-MS and IdeS digestion; Abstract), comprising:
loading a sample (The recombinant monoclonal IgG2/4 hybrid antibody (pI of peaks between 6.0 and 7.0; 1.1. Materials, pg. 50) having a protein of interest and at least one charge variant (intact antibody and its fragments after IdeS digestion, 1.2. Weak anion exchange chromatography and fraction collections, para. 3, pg. 50) of said protein of interest (The same instrument, column and mobile phases were used for the analysis of the antibody after IdeS digestion with a slightly modified gradient. 1.2. Weak anion exchange chromatography and fraction collections, para. 2, pg. 50) to an anion-exchange chromatography (AEX) column (A Waters Alliance HPLC and a weak anion exchange (WAX) column (ProPac WAX-10, 4.6 250 mm, Thermo Scientific, Sunnyvale, CA) were used for separation of antibody charge variants, 1.2. Weak anion exchange chromatography and fraction collections, para. 1, pg. 50) coupled to mass spectrometry (Under broadest reasonable interpretation, the claim limitation simply requires mass spectrometry to be conducted immediately after and does not require direct coupling; 2.2.2. Analysis of the collected fractions, pg. 51-55);
applying an increasing salt concentration gradient (Mobile phase B contained 10 mM Tris and 500 mM sodium chloride; 1.2. Weak anion exchange chromatography and fraction collections, para. 1, pg. 50) to the loaded anion exchange column to obtain an eluate (Samples were injected at 0% mobile phase B, after 5 min, mobile phase B was increased to 60% in 60 min. The column was then washed using 100% mobile phase B and then equilibrated using 0% mobile phase B for 10 min. 1.2. Weak anion exchange chromatography and fraction collections, para. 2, pg. 50);
collecting at least one fraction from b) (Fractions from analysis of both the intact antibody and its fragments after IdeS digestion were collected; 1.2. Weak anion exchange chromatography and fraction collections, para. 3, pg. 50); and
analyzing the at least one fraction to mass spectrometry analysis to characterize the at least one charge variant of said protein of interest (2.2.2. Analysis of the collected fractions, pg. 51-55).
Ponniah discloses the claimed invention as discussed above in claim 15. Ponniah discloses the protein of interest has a pI value ranges from 6.0-7.0 (The recombinant monoclonal IgG2/4 hybrid antibody (pI of peaks between 6.0 and 7.0; 1.1. Materials, pg. 50) and the charge variant is succinimide (1.5 Trypsin and Lys-C digestion, para. 3, page 50).
Ponniah teaches the range of 6.0-7.0, which overlaps with the claimed range 6.2-7.0. It would have been obvious to one of ordinary skill in the art before the effective filing date of the invention to have selected the overlapping portion of the ranges disclosed by the reference because selection of overlapping portion of ranges has been held to be a prima facie case of obviousness. See MPEP § 2144.05.I.
Ponniah does not disclose a step of deglycosylating a sample having a protein of interest and at least one charge variant. Ponniah also does not disclose the salt is ammonium salt in step c and using a AEX column coupled on-line to mass spectrometry.
Regarding a step of deglycosylating, Bobaly discloses a list of enzymatic and chemical sample preparation protocols for monoclonal antibodies (mAbs) for analytical characterization. Bobaly discloses that techniques such as N-deglycosylation using enzymes such as PNGase F (2.3. Introduction to N-deglycosylation, pg. 330-331) simplifies the sample and thus make the sample more suitable for chromatographic and mass spectrometric analysis (The analytical characterization of therapeutic monoclonal antibodies and related proteins usually incorporates various sample preparation methodologies. Indeed, quantitative and qualitative information can be enhanced by simplifying the sample, thanks to the removal of sources of heterogeneity (e.g. N-glycans) and/or by decreasing the molecular size of the tested protein by enzymatic or chemical fragmentation. These approaches make the sample more suitable for chromatographic and mass spectrometric analysis. Abstract).
In another analogous art, Promega discloses IdeS protease can be used with PNGase F within the same reaction to perform fragmentation and removal of Fc glycan in a single step (IdeS Protease can be used in the same reaction as PNGase F (Cat.# V4831) to perform fragmentation and removal of Fc glycans in a single step using the recommended digest buffer. Ten units of PNGase F may be sufficient to remove Fc glycans from 50µg of IgG in 2 hours at 37°C, although some optimization of PNGase F amount and incubation time may be required. Usage Information, page 2, Notes, bullet point 9, left column)
It would have been obvious to one of ordinary skill in the art to have incorporated a sample preparation step of N-deglycosylation to the method of Ponniah as suggested by Bobaly and Promega using PNGase F enzyme in a one pot reaction. One of ordinary skill in the art would be motivated to do so in order to improve mass spectrometry and chromatography results by simplifying the sample as disclosed by Bobaly (Abstract).
Regarding using AEX-coupled MS and an ammonium salt in step c, neither Ponniah, Promega nor Bobaly explicitly discloses the salt is ammonium salt. In an analogous art, Bardotti discloses LC-MS analysis using ionic eluent comprising ionic salt, in particular ammonium salt (Abstract). Bardotti discloses that a particular problem eluting analyte with an ionic eluent such as sodium buffer is that when coupled with MS, the sodim eluents can cause baseline noise and spiking in the mass spectrum (Bardotti, para. [0007]). Bardotti suggests using ionic eluent comprising ammonium salt can provide clean mass spectra and overcome the issue of sodium buffer (para. [0008]).
In another analogous art, Yan discloses a cation-exchange chromatography coupled to native mass spectrometry (Abstract) that can be reconfigured/repurposed for an anion-exchange coupled to mass spectrometry (Finally, this method may provide opportunities for further development of IEX-MS-based analysis of proteins, such as coupling of anion exchange chromatography (AEX) with online MS detection under similar conditions. This newly developed, versatile, and ultrasensitive native SCX-MS method represents a significant advancement for mAb charge heterogeneity analysis and therefore making it a valuable addition to our assay portfolio to support therapeutic mAb characterization. Conclusion). Yan discloses using conventional nonvolatile IEX buffers for direct mass spectrometry may be incompatible (Page 13014, left col., para. 2) and a strategy to address the issue is utilizing volatile salt (Page 13014, left col., para. 2).
Yan cited several instances of using ammonium-based salt/solution as pH and salt gradient for characterization of IdeS digestion (Leblanc et al. (14) reported a strong cation exchange (SCX)-MS method utilizing an ammonium formate- and ammonium acetate-based pH and salt gradient for characterization of IdeS digested mAbs. Another report by Muneeruddin et al. (15) described the use of an ammonium acetate-based salt gradient for WCX-MS analysis of proteins of molecular weights below ∼30 kDa. All of these previously reported IEX-MS methods showed successful separation and mass spectrometric detection; Page 13014, left col., para. 2). Yan specifically uses ammonium acetate and ammonium bicarbonate in their own experiment (Experimental Section). Yan specifies the need to optimize the pH and salt concentration changes to achieve efficient separation of the charge variatns while keeping allowing the mAbs to maintain their native states (In addition, the mobile phase composition and gradient were optimized to accommodate both pH and salt concentration changes to achieve efficient separation of the charge variants while keeping the pH (5.6–7.4) and salt concentration (20–150 mM) in a range where mAbs are maintained in native states. Results and Discussion, para. 1).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date to have derived an AEX-MS and replaced the sodium salt of Modified Ponniah with volatile ammonium salt as suggested by Bardotti and Yan. Doing so reduces baseline noise and spiking in the mass spectra as a result of coupling (Bardotti, para. [0007]-[0008]) while also maintaining the native state of the charge variants from pH changes (Yan, Results and Discussion, para. 1).
Regarding claim 16 and 17, Modified Ponniah discloses the claimed invention as discussed above in claim 15. Ponniah discloses the salt gradient is an elution gradient and ranges from about 0 mM to 300 mM sodium chloride (Mobile phase B contained 500 mM sodium chloride, and 60% mobile phase B is 300 mM sodium chloride; Samples were injected at 0% mobile phase B, after 5 min, mobile phase B was increased to 60% in 60 min. 1.2. Weak anion exchange chromatography and fraction collections, para. 2, pg. 50).
Ponniah teaches the range of 0 mM to 300 mM, which overlaps with the claimed range of 10 to 300 mM. It would have been obvious to one of ordinary skill in the art before the effective filing date of the invention to have selected the overlapping portion of the ranges disclosed by the reference because selection of overlapping portion of ranges has been held to be a prima facie case of obviousness. See MPEP § 2144.05.I.
Regarding claims 18-19, Modified Ponniah discloses the claimed invention as discussed above in claim 16 and 17 respectively. Ponniah (after incorporating ammonium salt) discloses the increasing salt concentration gradient applied is linear (After 5 min, a linear gradient from 10% mobile phase B to 60% mobile phase B within 50 min was applied to elute antibody variants from the column… The same instrument, column and mobile phases were used for the analysis of the antibody after IdeS digestion with a slightly modified gradient. 1.2. Weak anion exchange chromatography and fraction collections, para. 2-3, pg. 50).
Regarding claim 20, Modified Ponniah discloses the claimed invention as discussed above in claim 15. Ponniah discloses the method further comprising monitoring the eluate of b) for ultraviolet absorbance (The proteins eluting off the column were monitored using UV280 nm…Protein concentrations were determined using UV absorbance at 280 nm; 1.2. Weak anion exchange chromatography and fraction collections, para. 1 and 3, pg. 50) and collecting said at least one fraction that is eluted prior to or after elution of the protein of interest (Fractions from analysis of both the intact antibody and its fragments after IdeS digestion were collected; 1.2. Weak anion exchange chromatography and fraction collections, para. 3, pg. 50).
Regarding claim 23-24, Modified Ponniah discloses the claimed invention as discussed above in claim 15. Ponniah discloses the method further comprising digesting the sample prior to loading said sample on AEX column, wherein the sample is digested using IdeS or variant thereofs (The same instrument, column and mobile phases were used for the analysis of the antibody after IdeS digestion with a slightly modified gradient. 1.2. Weak anion exchange chromatography and fraction collections, para. 3, pg. 50).
Regarding claim 26, Ponniah discloses a method for characterizing at least one charge variant of a protein of interest (An efficient strategy to characterize recombinant monoclonal antibody charge variants was established using weak anion exchange chromatography, LC-MS and IdeS digestion; Abstract), comprising:
subjecting a sample (The recombinant monoclonal IgG2/4 hybrid antibody (pI of peaks between 6.0 and 7.0; 1.1. Materials, pg. 50) having a protein of interest and at least one charge variant (intact antibody and its fragments after IdeS digestion, 1.2. Weak anion exchange chromatography and fraction collections, para. 3, pg. 50) to digestion condition (The same instrument, column and mobile phases were used for the analysis of the antibody after IdeS digestion with a slightly modified gradient. 1.2. Weak anion exchange chromatography and fraction collections, para. 2, pg. 50) coupled to mass spectrometry (Under broadest reasonable interpretation, the claim limitation simply requires mass spectrometry to be conducted immediately after and does not require direct coupling; 2.2.2. Analysis of the collected fractions, pg. 51-55);
loading said sample to an anion-exchange chromatography (AEX) column (A Waters Alliance HPLC and a weak anion exchange (WAX) column (ProPac WAX-10, 4.6 250 mm, Thermo Scientific, Sunnyvale, CA) (The same instrument, column and mobile phases were used for the analysis of the antibody after IdeS digestion with a slightly modified gradient. 1.2. Weak anion exchange chromatography and fraction collections, para. 2, pg. 50) were used for separation of antibody charge variants, 1.2. Weak anion exchange chromatography and fraction collections, para. 1, pg. 50);
applying an increasing salt concentration gradient (Mobile phase B contained 10 mM Tris and 500 mM sodium chloride; 1.2. Weak anion exchange chromatography and fraction collections, para. 1, pg. 50) to the loaded anion exchange column to obtain an eluate (Samples were injected at 0% mobile phase B, after 5 min, mobile phase B was increased to 60% in 60 min. The column was then washed using 100% mobile phase B and then equilibrated using 0% mobile phase B for 10 min. 1.2. Weak anion exchange chromatography and fraction collections, para. 2, pg. 50);
collecting at least one fraction from b) (Fractions from analysis of both the intact antibody and its fragments after IdeS digestion were collected; 1.2. Weak anion exchange chromatography and fraction collections, para. 3, pg. 50); and
analyzing at least one fraction to mass spectrometry analysis to characterize the at least one charge variant of said protein of interest (2.2.2. Analysis of the collected fractions, pg. 51-55).
Ponniah discloses the claimed invention as discussed above in claim 26. Ponniah discloses the protein of interest has a pI value ranges from 6.0-7.0 (The recombinant monoclonal IgG2/4 hybrid antibody (pI of peaks between 6.0 and 7.0; 1.1. Materials, pg. 50) and the charge variant is succinimide (1.5 Trypsin and Lys-C digestion, para. 3, page 50).
Ponniah teaches the range of 6.0-7.0, which overlaps with the claimed range of 6.2-7.0. It would have been obvious to one of ordinary skill in the art before the effective filing date of the invention to have selected the overlapping portion of the ranges disclosed by the reference because selection of overlapping portion of ranges has been held to be a prima facie case of obviousness. See MPEP § 2144.05.I.
Ponniah does not disclose a step of digesting and deglycosylating a sample having a protein of interest and at least one charge variant. Furthermore, Ponniah does not disclose the salt is ammonium salt and using a AEX column coupled on-line to mass spectrometry.
Regarding a step of deglycosylating and digeting, Bobaly discloses a list of enzymatic and chemical sample preparation protocols for monoclonal antibodies (mAbs) for analytical characterization. Bobaly discloses that techniques such as N-deglycosylation using enzymes such as PNGase F (2.3. Introduction to N-deglycosylation, pg. 330-331) simplifies the sample and thus make the sample more suitable for chromatographic and mass spectrometric analysis (The analytical characterization of therapeutic monoclonal antibodies and related proteins usually incorporates various sample preparation methodologies. Indeed, quantitative and qualitative information can be enhanced by simplifying the sample, thanks to the removal of sources of heterogeneity (e.g. N-glycans) and/or by decreasing the molecular size of the tested protein by enzymatic or chemical fragmentation. These approaches make the sample more suitable for chromatographic and mass spectrometric analysis. Abstract).
In another analogous art, Promega discloses IdeS protease can be used with PNGase F within the same reaction to perform fragmentation and removal of Fc glycan in a single step (IdeS Protease can be used in the same reaction as PNGase F (Cat.# V4831) to perform fragmentation and removal of Fc glycans in a single step using the recommended digest buffer. Ten units of PNGase F may be sufficient to remove Fc glycans from 50µg of IgG in 2 hours at 37°C, although some optimization of PNGase F amount and incubation time may be required. Usage Information, page 2, Notes, bullet point 9, left column)
It would have been obvious to one of ordinary skill in the art to have incorporated a sample preparation step of N-deglycosylation to the method of Ponniah as suggested by Bobaly and Promega using PNGase F enzyme in a one pot reaction. One of ordinary skill in the art would be motivated to do so in order to improve mass spectrometry and chromatography results by simplifying the sample as disclosed by Bobaly (Abstract).
Regarding using an AEX column coupled to spectrometry and ammonium salt in step c, neither Ponniah, Promega nor Bobaly explicitly discloses the salt is ammonium salt. In an analogous art, Bardotti discloses LC-MS analysis using ionic eluent comprising ionic salt, in particular ammonium salt (Abstract). Bardotti discloses that a particular problem eluting analyte with an ionic eluent such as sodium buffer is that when coupled with MS, the sodim eluents can cause baseline noise and spiking in the mass spectrum (Bardotti, para. [0007]). Bardotti suggests using ionic eluent comprising ammonium salt can provide clean mass spectra and overcome the issue of sodium buffer (para. [0008]).
In another analogous art, Yan discloses a cation-exchange chromatography coupled to native mass spectrometry (Abstract) that can be reconfigured/repurposed for an anion-exchange coupled to mass spectrometry (Finally, this method may provide opportunities for further development of IEX-MS-based analysis of proteins, such as coupling of anion exchange chromatography (AEX) with online MS detection under similar conditions. This newly developed, versatile, and ultrasensitive native SCX-MS method represents a significant advancement for mAb charge heterogeneity analysis and therefore making it a valuable addition to our assay portfolio to support therapeutic mAb characterization. Conclusion). Yan discloses using conventional nonvolatile IEX buffers for direct mass spectrometry may be incompatible (Page 13014, left col., para. 2) and a strategy to address the issue is utilizing volatile salt (Page 13014, left col., para. 2).
Yan cited several instances of using ammonium-based salt/solution as pH and salt gradient for characterization of IdeS digestion (Leblanc et al. (14) reported a strong cation exchange (SCX)-MS method utilizing an ammonium formate- and ammonium acetate-based pH and salt gradient for characterization of IdeS digested mAbs. Another report by Muneeruddin et al. (15) described the use of an ammonium acetate-based salt gradient for WCX-MS analysis of proteins of molecular weights below ∼30 kDa. All of these previously reported IEX-MS methods showed successful separation and mass spectrometric detection; Page 13014, left col., para. 2). Yan specifically uses ammonium acetate and ammonium bicarbonate in their own experiment (Experimental Section). Yan specifies the need to optimize the pH and salt concentration changes to achieve efficient separation of the charge variatns while keeping allowing the mAbs to maintain their native states (In addition, the mobile phase composition and gradient were optimized to accommodate both pH and salt concentration changes to achieve efficient separation of the charge variants while keeping the pH (5.6–7.4) and salt concentration (20–150 mM) in a range where mAbs are maintained in native states. Results and Discussion, para. 1).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date to have derived an AEX-MS and replaced the sodium salt of Ponniah with volatile ammonium salt as suggested by Bardotti and Yan. Doing so reduces baseline noise and spiking in the mass spectra as a result of coupling (Bardotti, para. [0007]-[0008]) while also maintaining the native state of the charge variants from pH changes (Yan, Results and Discussion, para. 1).
Regarding claim 28, Modified Ponniah discloses the claimed invention as discussed above in claim 26. Ponniah discloses the sample is digested using IdeS or variant thereofs (The same instrument, column and mobile phases were used for the analysis of the antibody after IdeS digestion with a slightly modified gradient. 1.2. Weak anion exchange chromatography and fraction collections, para. 3, pg. 50).
Regarding claim 29 and 30, Modified Ponniah discloses the claimed invention as discussed above in claim 26. Modified Ponniah discloses the salt gradient is an elution gradient and ranges from about 0 mM to 300 mM sodium chloride (Mobile phase B contained 500 mM sodium chloride, and 60% mobile phase B is 300 mM sodium chloride; Samples were injected at 0% mobile phase B, after 5 min, mobile phase B was increased to 60% in 60 min. 1.2. Weak anion exchange chromatography and fraction collections, para. 2, pg. 50).
Ponniah teaches the range of 0 mM to 300 mM, which overlaps with the claimed range of 10 to 300 mM. It would have been obvious to one of ordinary skill in the art before the effective filing date of the invention to have selected the overlapping portion of the ranges disclosed by the reference because selection of overlapping portion of ranges has been held to be a prima facie case of obviousness. See MPEP § 2144.05.I.
Regarding claim 31, Modified Ponniah discloses the claimed invention as discussed above in claim 26. Ponniah discloses the increasing salt concentration gradient applied is linear (After 5 min, a linear gradient from 10% mobile phase B to 60% mobile phase B within 50 min was applied to elute antibody variants from the column… The same instrument, column and mobile phases were used for the analysis of the antibody after IdeS digestion with a slightly modified gradient. 1.2. Weak anion exchange chromatography and fraction collections, para. 2-3, pg. 50).
Regarding claim 32, Modified Ponniah discloses the claimed invention as discussed above in claim 30. Ponniah discloses the increasing salt concentration gradient applied is linear (After 5 min, a linear gradient from 10% mobile phase B to 60% mobile phase B within 50 min was applied to elute antibody variants from the column… The same instrument, column and mobile phases were used for the analysis of the antibody after IdeS digestion with a slightly modified gradient. 1.2. Weak anion exchange chromatography and fraction collections, para. 2-3, pg. 50) (Note: It is presumed linear gradient is also applied to the analysis of the antibody after IdeS digestion).
Regarding claim 33, Modified Ponniah discloses the claimed invention as discussed above in claim 26. Ponniah discloses the method further comprising monitoring the eluate of b) for ultraviolet absorbance (The proteins eluting off the column were monitored using UV280 nm…Protein concentrations were determined using UV absorbance at 280 nm; 1.2. Weak anion exchange chromatography and fraction collections, para. 1 and 3, pg. 50) and collecting the at least one fraction that is eluted prior to or after elution of the protein of interest (Fractions from analysis of both the intact antibody and its fragments after IdeS digestion were collected; 1.2. Weak anion exchange chromatography and fraction collections, para. 3, pg. 50).
Regarding claim 36, Modified Ponniah discloses the claimed invention as discussed above in claim 26. Ponniah discloses the method further comprising digesting the sample prior to loading said sample on AEX column, wherein the digestion conditions include use of IdeS or variant thereofs (The same instrument, column and mobile phases were used for the analysis of the antibody after IdeS digestion with a slightly modified gradient. 1.2. Weak anion exchange chromatography and fraction collections, para. 3, pg. 50).
Claim(s) 25 is/are rejected under 35 U.S.C. 103 as being unpatentable over Ponniah in view of Yan, Bardotti, Bobaly and Promega as applied to claim 15 above, and further in view of Bailey.
Regarding claim 25, Modified Ponniah discloses the claimed invention as discussed above in claim 15. Neither Ponniah, Yan, Bardotti, Bobaly, nor Prometa explicitly discloses running a mass spectrometry under native condition.
In an analogous art, Bailey discloses a procedure to perform the analysis of monoclonal antibodies using native MS (charge variant native mass spectrometry) for characterization of variants such as deamidation (Characterization of variants such as deamidation, which are traditionally unattainable by intact mass due to their minimal molecular weight differences, were measured unambiguously by mass and retention time to allow confident MS1 identification. We demonstrate that efficient chromatographic separation allows introduction of the purified forms of the charge variant isoforms into the Orbitrap mass spectrometer. Abstract).
It would have been obvious to one of ordinary skill in the art before the effective filing date to used Orbitrap MS system (which comes with native MS setting) disclosed by Bailey to the method of Modified Ponniah. Doing so increase baseline resolution for measurement of complex biological compound such as mAbs (Introduction, para. 2, pg. 1214, Bailey).
Claim(s) 27 is/are rejected under 35 U.S.C. 103 as being unpatentable over Ponniah in view of Yan, Bardotti, Bobaly and Promega as applied to claim 26 above, and further in view of Bailey.
Regarding claim 27, Modified Ponniah discloses the claimed invention as discussed above in claim 26. Neither Ponniah, Yan, Bardotti, Bobaly, nor Prometa explicitly discloses running a mass spectrometry under native condition.
In an analogous art, Bailey discloses a procedure to perform the analysis of monoclonal antibodies using native MS (charge variant native mass spectrometry) for characterization of variants such as deamidation (Characterization of variants such as deamidation, which are traditionally unattainable by intact mass due to their minimal molecular weight differences, were measured unambiguously by mass and retention time to allow confident MS1 identification. We demonstrate that efficient chromatographic separation allows introduction of the purified forms of the charge variant isoforms into the Orbitrap mass spectrometer. Abstract).
It would have been obvious to one of ordinary skill in the art before the effective filing date to used Orbitrap MS system (which comes with native MS setting) disclosed by Bailey to the method of Modified Ponniah. Doing so increases baseline resolution for measurement of complex biological compound such as mAbs (Introduction, para. 2, pg. 1214, Bailey).
Conclusion
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure.
US 20150285771 A1 discloses methods for analyzing compositions of polypeptides such as antibodies by ionic strength-mediated pH gradient ion exchange chromatography.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/M.H./ Examiner, Art Unit 1758
/MARIS R KESSEL/ Supervisory Patent Examiner, Art Unit 1758
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